Immunological assays are receiving attention as a method of identifying and quantitating, with high accuracy, proteins such as virus, bacteria, and allergenic substance contained in biological samples (for example, blood).
The immunological assay is roughly classified into an immunonephelometry and an immunolabeling assay. The immunonephelometry is a method of determining the change in turbidity of a sample solution that is caused by an antigen-antibody complex produced by an antigen-antibody reaction. The immunolabeling assay is a method of determining the change in the amount of a labeling substance after an antigen-antibody reaction by using an antibody labeled with the labeling substance.
The immunolabeling assay is subdivided according to the type of the labeling substance. Examples thereof include a radioimmunoassay in which a radioisotope is used as the labeling substance, an enzyme immunoassay (EIA) in which an enzyme is used, and a fluorescence immunoassay in which a fluorescent substance is used. In the EIA, as compared to other immunolabeling assays, the safety of the labeling substance is higher, it can be carried out by a simpler operation, and the measurement accuracy is higher. Therefore the EIA is used frequently.
A typical example of the EIA is an enzyme-linked immunosorbent assay (ELISA). An example of the ELISA is described with reference to FIG. 1.
<Step 1-A: Forming a Solid-Phase Antigen>
A solution containing a capture antigen having an epitope identical to that of an antigen to be measured is introduced into a reaction chamber 7 and is maintained at a predetermined temperature for a predetermined period of time, and thereby the capture antigen is allowed to adsorb to the surface of the reaction chamber 7. Thereafter the capture antigen to be allowed to adsorb is covered with protein that is not involved in a later antigen-antibody reaction and enzyme reaction (blocking). Thus a solid-phase antigen 6 is formed inside the reaction chamber 7. The solid-phase antigen 6 is being fixed to the surface of the reaction chamber 7 and therefore is not removed from the reaction chamber 7 by the washing described later.
<Step 1-B: Binding Reactions Between Antibody and Antigen to be Measured as Well as Solid-Phase Antigen>
An antibody 3 that specifically binds to an antigen to be measured 2 is added to a sample solution 9. The antibody 3 is being labeled with a labeling substance (for example, an enzyme) 4. Thereafter, the sample solution 9 containing the antibody 3 is introduced into the reaction chamber 7. Thus, the antigen-antibody reaction proceeds between the antibody 3 and the antigen to be measured 2 as well as the solid-phase antigen 6 in the reaction chamber 7.
<Step 1-C: Removal of Unreacted Antibody and Antigen-Antibody Complex>
Using a wash solution 10 typified by Tris-HCl buffer, the inside of the reaction chamber 7 is washed. Thereby the antigen-antibody complex formed through binding of the antibody 3 to the antigen to be measured 2 and the antibody 3 that has not been bound to the solid-phase antigen 6 are removed from the reaction chamber 7. Accordingly, a conjugate of the solid-phase antigen 6 and the antibody 3 remains in the reaction chamber 7.
Subsequently, the amount of the labeling substance 4 of the conjugate of the solid-phase antigen 6 and the antibody 3 that has remained in the reaction chamber 7 is measured. This measurement is carried out, for example, as follows. First, a solution containing a measuring reagent (for example, a substrate of the enzyme) that reacts with the labeling substance 4 is prepared. Next, this solution is introduced into the reaction chamber 7, and thereby a measurement solution is obtained that contains the measuring reagent and the conjugate of the solid-phase antigen 6 and the antibody 3. After the reaction between the measuring reagent and the labeling substance 4 is allowed to proceed in the measurement solution, a signal that reflects the amount of reaction product is detected.
As a result of this measurement, the amount of the antigen to be measured 2 in the sample solution 9 is calculated based on the amount of the solid-phase antigen 6 and the amount of the sample solution 9 introduced into the reaction chamber 7 in Step 1-B.
From the viewpoint of measuring the amount of the substance to be measured in the sample solution using a trace amount of sample solution in a short period of time, a chip-type biosensor is receiving attention. For example, JP 2 (1990)-062952 A discloses a chip-type biosensor including an insulating chip substrate, an electrode system disposed on the chip substrate, an enzyme reaction layer disposed on the electrode system, and an insulating layer that has notches and is disposed above the chip substrate in such a manner that the electrode system and the enzyme reaction layer are exposed. The enzyme reaction layer contains a measuring reagent for inducing an enzymatic cycling reaction, which is typified by an oxidoreductase and an electron mediator. An enzyme containing as a substrate the substance to be measured is used as the oxidoreductase. For example, glucose oxidase is used when the glucose amount is to be measured, and cholesterol oxidase is used when the cholesterol amount is to be measured. In this biosensor, a sample solution is dripped into the notches, so that the measuring reagent is dissolved in the sample solution. Thereby the reaction between the enzyme and the substance to be measured through an electron mediator (an enzymatic cycling reaction) proceeds. The amount of the substance to be measured in the sample solution is calculated based on the oxidation current value that is obtained by electrochemically oxidizing the electron mediator reduced by the enzyme reaction.
In the conventional immunological assay as shown in FIG. 1, as described above, Step 1-C is carried out using a wash solution typified by Tris-HCl buffer. Accordingly, in order to carry out the immunological assay on a chip, it is necessary to supply the wash solution from the outside of the chip or to allow the wash solution to be retained on the chip beforehand. However, when the wash solution is supplied from the outside of the chip, extra time and effort is required for the supply. When the wash solution is allowed to be retained on the chip, this solution desirably is allowed to be retained on the chip in a hermetic state and in the state that facilitates it to be introduced into the reaction chamber for washing. However, it is not easy to form such a retaining state on a chip.